Antiviral, anti-inflammatory and antioxidant effects of curcumin and curcuminoids in SH-SY5Y cells infected by SARS-CoV-2

COVID-19, caused by SARS-CoV-2, affects neuronal cells, causing several symptoms such as memory loss, anosmia and brain inflammation. Curcuminoids (Me08 e Me23) and curcumin (CUR) are derived from Curcuma Longa extract (EXT). Many therapeutic actions have been linked to these compounds, including antiviral action. Given the severe implications of COVID-19, especially within the central nervous system, our study aims to shed light on the therapeutic potential of curcuminoids against SARS-CoV-2 infection, particularly in neuronal cells. Here, we investigated the effects of CUR, EXT, Me08 and Me23 in human neuroblastoma SH-SY5Y. We observed that Me23 significantly decreased the expression of plasma membrane-associated transmembrane protease serine 2 (TMPRSS2) and TMPRSS11D, consequently mitigating the elevated ROS levels induced by SARS-CoV-2. Furthermore, Me23 exhibited antioxidative properties by increasing NRF2 gene expression and restoring NQO1 activity following SARS-CoV-2 infection. Both Me08 and Me23 effectively reduced SARS-CoV-2 replication in SH-SY5Y cells overexpressing ACE2 (SH-ACE2). Additionally, all of these compounds demonstrated the ability to decrease proinflammatory cytokines such as IL-6, TNF-α, and IL-17, while Me08 specifically reduced INF-γ levels. Our findings suggest that curcuminoid Me23 could serve as a potential agent for mitigating the impact of COVID-19, particularly within the context of central nervous system involvement.


Cytotoxicity of CUR, EXT and curcuminoids on SH-SY5Y cells
To investigate the cytotoxic effects of CUR, EXT (Extract) and curcuminoids on SH-SY5Y cells, we treated the cells with different concentrations of the compounds and performed a MTT assay after 24 h.We observed that CUR exhibited cytotoxicity at concentrations of 14, 18, 28, and 90 µg/mL, resulting in cell viabilities of 40.1%, 40.88%, 33.4%, and 20.46%, respectively (Fig. 1A,B, One-way ANOVA, F 6,31 = 35.72,p < 0.0001).Conversely, the viability of SH-SY5Y cells remained at or above 95% when exposed to CUR concentrations of 7.2 µg/mL or lower (Fig. 1A,B).Consequently, we decided to use the non-toxic higher concentration of CUR, which was 7.2 µg/mL, in our future experiments.In contrast, EXT did not exhibit cytotoxicity at any of the concentrations examined.We chose the concentration of 3.6 µg/mL, the highest concentration that maintained viability similar to the control (CTL) (Fig. 1C), for our subsequent experiments.Regarding the curcuminoids Me08 and Me23, no toxicity

ME23 reduces ROS induced by SARS-CoV-2
To assess the potential of the compounds to mitigate ROS production following SARS-CoV-2 infection in SH-SY5Y cells, we infected the cells with the virus.After a 2-h incubation with SARS-CoV-2, we removed the virus and allowed the cells to incubate for an additional 24 h only with media (Fig. 3A).Basal ROS levels were quantified in MOCK cells, which served as the reference set at 100%.Cells infected with SARS-CoV-2 but left untreated were designated as the CTL.

Me23 demonstrates antioxidant effects following SARS-CoV-2 infection
Based on previous findings indicating the antioxidant effects of CUR or curcuminoids via the NRF2 pathway, we decided to investigate the expression of this gene following SARS-CoV-2 infection and treatment with Me23, the only compound capable of reducing ROS levels (Fig. 3B).Notably, SARS-CoV-2 infection did not change the gene expression of NRF2.However, treatment with Me23 resulted in an approximately tenfold increase in NRF2 expression compared to the CTL and MOCK groups (Fig. 4A, One-way ANOVA, F 2,13 = 10.23,p = 0.0021).Since NRF2 upregulates NQO1, we also assessed NQO1 activity.SARS-CoV-2 infection reduced NQO1 activity by 1.7-fold (Fig. 4B, One-way ANOVA, F 5,25 = 8.440, p = 0.0045, however, in the presence of Me23, the activity was restored to levels similar to those observed in the MOCK.

ACE2 Overexpression in SH-SY5Y Cells reveal the antiviral effects of Curcuminoids Me08 and Me23
Considering previous publications indicating that SH-SY5Y cells exhibit low levels of SARS-CoV-2 replication 14 , we have decided to overexpress ACE2 to bolster viral replication within these cells.To achieve this, we used a lentivector carrying the ACE2 gene, successfully establishing the SH-ACE2 cell line.SH-SY5Y and SH-ACE2 cells were treated with CUR, EXT or curcuminoids (Me08 and Me23) for 2 h, after the treatment removal, the cells were infected with SARS-CoV-2 for 2 h.The virus was removed, and the RT-qPCR was performed after 24 h (Fig. 5A).Our findings revealed an upregulation of ACE2 expression in SH-ACE2, with levels exceeding 10,000 times that of SH-SY5Y cells (Fig. 5B, Unpaired t-test, t 4 = 4.758, p < 0.05).This substantial ACE2 overexpression in SH-ACE2 corresponded with a significantly higher viral load, reaching 1.1X10 8 PFU/mL at 24 h post-infection, representing a 4-log increase when compared to SH-SY5Y (Fig. 5C, One-way ANOVA, F 9,39 = 123.4,p < 0.0001).

CUR, EXT and curcuminoids ME08 and ME23 reduces the levels of INF-γ, TNF-α, IL-6 and IL-17 in SH-ACE2 cells infected with SARS-CoV-2
In light of the recognized role of inflammation in neuronal cell death and the potential neurotoxicity of various pro-inflammatory factors, including INF-γ, TNF-α, IL-6 and IL-17 44,45 , we sought to investigate the impact of CUR, EXT, and curcuminoids ME08 and ME23 on these factors in SH-ACE2 cells following SARS-CoV-2 infection.
To assess their effect on pro-inflammatory protein expression, we subjected SH-ACE2 cells to pretreatment with these compounds for 2 h.Subsequently, the cells were exposed to SARS-CoV-2 for 2 h, followed by the collection of supernatant after 24 h.We then quantified pro-inflammatory factors via ELISA (Fig. 6A).

Discussion
Studies investigating the utility and efficacy of CUR curcuminoids in various medical conditions, particularly in the context of COVID-19, have been gaining momentum during the ongoing pandemic.Recent clinical trials have yielded promising results, demonstrating the beneficial impact of CUR and curcuminoids on COVID-19.These effects encompass a reduction in time of hospitalization 46 , modulation of elevated inflammatory cytokine levels 47 , and expedited symptomatic recovery 48 .Understanding the devastating consequences of COVID-19,  mainly on the central nervous system, we aimed to investigate the effects of CUR, EXT, and curcuminoids on neuronal cell line.
In our study we used CUR, obtained from a nutritional supplement capsule, EXT (Curcuma longa) and the curcuminoids, Me08 and Me23.We observed that concentrations of CUR exceeding 7.2 µg/mL exhibited cytotoxic effects in SH-SY5Y, reducing cell viability to less than 50%.In contrast, EXT and Me08 demonstrated no cytotoxicity within the concentrations studied.However, Me23 displayed cytotoxicity at concentrations above 60 µM.As a result, we opted to utilize CUR at 7.2 µg/mL, EXT at 3.6 µg/mL, and Me08 and Me23 at 60 µM.2021) used higher concentrations of CUR and EXT in their studies 49,50 .Nevertheless, it's important to note that they used VERO-E6 cells, a monkey cell line commonly used in SARS-CoV-2 replication studies, and also observed cytotoxic effects in the cells.
Once defined the non-cytotoxic concentrations of the compounds we have decided to investigate the possible effects of CUR, EXT and curcuminoids (Me08 and Me23) on the expression of key host receptors for the virus.A previous in silico study suggested that CUR may interact with the spike glycoprotein of SARS-CoV-2, impeding the virus-receptor interaction by binding to ACE2 residues 51 .
Our results did not reveal significant modulation in the gene or protein expression of ACE2 or furin.However, curcuminoids Me23 and Me08 demonstrated reductions in protease levels.Specifically, Me08 decreased the protein levels of TMPRSS11D, while Me23 reduced the gene expression of TMPRSS11D and the protein level of TMPRSS2.Recent studies have associated TMPRSS11D with enhanced SARS-CoV-2 entry 15,17 .Notably, TMPRSS11D is expressed in brain tissues (https:// www.prote inatl as.org/ ENSG0 00001 53802-TMPRS S11D/ brain, accessed on 15 October 2023), and overexpression of this protease in VERO-E6 cells has been shown to enhance SARS-CoV-2 entry 17 .Previous research has indicated that CUR decreased TMPRSS2 expression in prostate cancer cells, although this effect was not observed in SH-SY5Y cells 52 .It's also worth noting that Me23 is a newly explored curcuminoid that has not been tested in the context of COVID-19 until now.
Given the previous studies highlighting the antioxidative properties of CUR, EXT, and curcuminoids, we decided to investigate the levels of ROS following treatment with these compounds and SARS-CoV-2 infection.Our findings revealed that SARS-CoV-2 infection led to an increase in ROS levels in SH-SY5Y cells.This observation aligns with existing literature, as severe cases of COVID-19, where the virus penetrates the central  www.nature.com/scientificreports/nervous system (CNS), can trigger a cytokine storm.This, in turn, results in heightened ROS production and oxidative stress (OS).In response to OS, inflammatory cells release more pro-inflammatory cytokines, thereby exacerbating inflammation and intensifying ROS and OS.This creates a detrimental cycle of events [53][54][55] .These pro-inflammatory cytokines and OS can contribute to demyelination and axonal damage.Only Me23 reduced the levels of ROS to the MOCK levels.Curcumin's ability to cross the blood-brain barrier (BBB) is of particular interest since it can potentially reduce elevated ROS levels, protect the brain from lipid peroxidation, and decrease neuronal death due to oxidative damage 35 .Additional studies have observed a reduction in ROS levels following the administration of curcuminoids 56,57 , highlighting the antioxidative potential of these compounds.Contradictory results in our study might be attributed to differences in purity levels, concentration measurements, and the use of CUR dietary supplements.We also decided to investigate the NRF2 expression, responsible for the body's antioxidant response 58 .A study using primary mouse osteoclast cultures revealed that treatment with pure CUR increased the expression of the NRF-2 gene 58 .NRF2 plays a crucial role in maintaining cellular homeostasis by regulating the expression of antioxidant enzymes and elements in the antioxidant response during oxidative stress (OS) processes 59 , such as heme oxygenase (HO-1) and NQO1, which mitigate OS progression and maintain redox balance, particularly in injured tissues and organic failure 60 .During infection, the SARS-CoV-2 can inhibit the NRF2 pathway, because the virus causes an exacerbated inflammatory response that contributes to the decrease of the NRF2 activity 60 , therefore, the virus causes an increase in OS and inflammatory disorder 61 .Ryan et al., 2022, observed that NRF2 can inhibit not only Interferon b but also IL-6 and TNF in inflammatory macrophages 62 .A clinical trial analyzed children with COVID-19 and observed a decrease in NRF2 levels in the children with COVID-19 when compared to the control group 63 .Some flavonoids e.g., curcumin, resveratrol, and naringenin, can decrease the OS through the NRF2 pathway 60 .Supporting this, Ding et al., 2022, analyzed a high-fat diet-fed mouse model treated with bisdemethoxycurcumin, observing increased NRF2 protein expression in the treated group compared with the control group.Additionally, in a study treating mice with traumatic brain injury with curcumin, an increase in both NRF2 and NQO-1 protein expression was observed compared to the control group 64 .
Our study aligns with these findings; we observed that the Me23 compound increased NRF2 gene expression in SH-SY5Y cells infected with SARS-CoV-2 compared with the control group.Additionally, we measured NQO1 activity and found that SARS-CoV-2 infection reduced NQO1 activity.However, in the presence of Me23, the activity was restored to levels similar to those observed in the MOCK.These results support the antioxidant role of curcuminoids such as Me23.
We further investigated the effects of CUR, EXT, and curcuminoids on SARS-CoV-2 replication.Previous studies involving SARS-CoV-2 infection in SH-SY5Y cells had shown limited viral replication 14 .To address this, we have decided to overexpress the ACE2 receptor in SH-SY5Y cells, thereby enhancing their capacity for extensive SARS-CoV-2 replication.Indeed, this genetic modification resulted in a viral load in SH-ACE2 cells that was 10,000 times higher than that in SH-SY5Y cells.Notably, the overexpression of the ACE2 receptor in SH-SY5Y cells revealed an antiviral effect of Me08 and Me23.It's worth mentioning that the antiviral effect of Me23 appeared to be more pronounced than that of Me08.
It has been demonstrated a reduction in viral replication in Calu-3 cells (human lung cancer cells) following treatment with EXT, CUR (in the form of a nutritional supplement capsule), and pure CUR 49 .They also reported a decrease in viral load in SARS-CoV-2-infected Vero-E6 cells when exposed to various concentrations of pure CUR.However, it's important to note that the authors used a concentration that was 10 times higher than the one we employed, and this higher concentration exhibited toxicity in our cells.To the best of our knowledge our study is the first to evaluate the antiviral effects of CUR, EXT and curcuminoids in neuronal cells.
Given the well-established anti-inflammatory properties of curcuminoids 40,65 , we further explored their role in modulating cytokines, including IL-6, IL-17, IFN-γ, and TNF-α, which are often elevated in COVID-19.Findings from other studies have shown increased cytokine levels in patients with neurological syndromes and COVID-19 22,24 .Interestingly, treatment with curcumin led to decreased gene expression of these inflammatory cytokines in peripheral blood mononuclear cells 50 .In alignment with these findings, our study indicated that all compounds (CUR, EXT, Me08, and Me23) reduced concentrations of IL-6, IL-17, and TNF-α, while Me08 specifically reduced IFN-γ levels in SH-ACE2 cells.

Conclusion
As summarized in Fig. 7, our study has revealed promising findings regarding the effects of curcuminoid Me23.We observed a reduction in TMPRSS2 and TMPRSS11D expression, a decrease in ROS levels induced by SARS-CoV-2, increase of the antioxidative pathway by modulation of NRF2 and NQO1, inhibition of SARS-CoV-2 replication, and noted anti-inflammatory effects in neuronal cells.These results not only contribute valuable insights into the potential of curcuminoids but also underscore the promising therapeutic prospects offered by Me23 in the context of COVID-19 treatment.
In conclusion, our study suggests that curcuminoid Me23 holds significant promise as an agent for mitigating the impact of COVID-19, particularly in the context of CNS involvement.Further research is warranted to delve deeper into its mechanisms of action and to assess its efficacy in clinical settings.These findings reinforce the importance of investigating natural compounds as potential allies in the battle against COVID-19 and related neurological complications.

Viral production and infection
Viral production was conducted in VERO-E6 cells (African green monkey kidney cells, ATCC CRL-1586), as previously described 66  www.nature.com/scientificreports/without phenol red, supplemented with NaHCO 3 , 10% fetal bovine serum, and 0.02 mg/mL of gentamicin (Sigma Aldrich), hereafter referred to as cDMEM.The cells were maintained in a humid incubator at 37 °C in an atmosphere of 5% CO 2 .For plating, they were trypsinized and seeded in 24-well plates for the infection and treatment protocols.All in vitro experiments involving the propagation, titration, and infection of SARS-CoV-2 were carried out in the Biosafety Level III Laboratory (NB III) at the Biological Institute, in collaboration with Professor Dr. Líria Hiromi Okuda, following the regulatory recommendations of PAHO and WHO.The infection of SH-SY5Y cells followed the protocol previously described 16 .In summary, the cells were infected with SARS-CoV-2 isolated from a Brazilian patient (EPI_ISL_413016), using a multiplicity of infection (MOI) of 0.2 for 2 h at 37 °C.After infection, the cells were washed with PBS, and 500 µL of culture medium was added.After 24 h of infection, the viral supernatant was collected for RNA extraction, and the cells also had their total RNA extracted.

Overexpression model of ACE2 in the SH-SY5Y cell line
The ACE2 overexpression model was developed in the SH-SY5Y cell line.SH-SY5Y cells were transduced with a lentiviral vector to overexpress ACE2 (pLENTI-hACE2-HygR, Addgene #161758).After 48 h of transfection, the cells were cultured in cDMEM for expansion and selection with hygromycin (80 μg/mL) for 14 days.Following this selection period, the cells were frozen for further experiments.

RNA extraction and RT-qPCR
The gene expression of ACE2, TMPRSS2, TMPRSS11D and Furin, was assessed using RT-qPCR.Firstly, total RNA from the cells was extracted using Trizol reagent (Invitrogen) following the manufacturer's protocol.The purity and concentration of RNA were determined by absorbance using the NanoVue instrument (GE Healthcare).For the reverse transcription of RNA into cDNA, the High Capacity Kit (Applied Biosystems) was used.
The reaction mixture consisted of 2 μg of RNA, 2 μL of 10X RT Buffer, 0.8 μL of 25X dNTP Mix (100 mM), 2 μL of 10X RT Random Primers, and 1 μL of the enzyme, and the volume was made up with water to achieve a final volume of 20 μL.The reaction conditions were as follows: 25 °C/10 min, 37 °C/120 min, and 85 °C/5 min.

Figure 2 .
Figure 2. SARS-CoV-2 receptors expression in SH-SY5Y.Cells were treated with CUR, EXT, Me08 or Me23 for 24 h and the gene expression was analyzed by RT-qPCR and protein expression was analyzed by Western-blotting.Gene expression of (A) ACE2 (B) Furin, (C) TMPRSS2, (D) TMPRSS11D.Gene expression was normalized by the endogenous expression of RPL35.(E) Protein expression according to western blot analyses.(F) Histograms reporting the mean ± SD of (G) ACE2, (H) TMPRSS2, (I) TMPRSS11D levels after normalization with the average intensity of the bands from four independent experiments.Protein expression was normalized by the endogenous expression of GAPDH.CTL: cells without treatment, CUR: curcumin, EXT: extract.*p < 0.05.(n = 4-13).The entire blots are presented in Supplementary Figure S1.

Figure 3 .
Figure 3. ROS quantification after treatment with CUR, EXT, Me08, Me23.Cells were treated with CUR, EXT, Me08 or Me23 for 2 h, after, they were infected with SARS-CoV-2 (MOI 0.2) for 24 h and the ROS was measured by flow cytometry.The percentage of fluorescence was compared to the MOCK.*p < 0.05.(n = 4-6).The schematic diagram illustrating the infection process was based on a previous publication 67 .
. The cells were cultured in 10 cm plates with DMEM (Dulbecco's Modified Eagle's)